In a developing multicellular organism, the formation of the anteroposterior (AP), dorsoventral (DV), and left-right (LR) body axes is a critical early step in the formation of the body plan. Members of the Wnt family of secreted signaling molecules are important participants in the specification of these body axes. Eighteen Wnt genes have been identified in the mouse and human genomes and many are co-expressed during embryogenesis begging the question of how specificity arises. One way specificity could be achieved is through the selective activation of different signal transduction pathways. One well-supported pathway, the canonical Wnt/beta-catenin pathway, signals through beta-catenin, while other alternative signaling pathways, known as the Wnt/planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway are less well-defined and utilize different signal transducers. The main goals of this project are to understand how Wnts participate in the formation and elaboration of the body plan during early mouse development, and the molecular pathways through which these Wnts signal. Wnts, beta-catenin, and the specification of the LR body axis The alignment of the LR body axis relative to the AP and DV body axes is central to the organization of the vertebrate body plan and is controlled by the node/organizer. Somitogenesis plays a key role in embryo morphogenesis as a principal component of AP elongation. How morphogenesis is coupled to axis specification is not well understood. Wnt3a regulates somitogenesis by controlling the segmentation clock and the molecular oscillations of the Notch pathway. We have demonstrated that Wnt3a, signaling through beta-catenin, is required for LR asymmetry and is upstream of left-determining, and asymmetrically expressed, genes in the node. Wnt3a regulates both the activation of the left determinant, Nodal, in the node via the Delta/Notch pathway, as well as the symmetry-breaking activity of mechanosensory cilia. Thus Wnt3a links the segmentation clock and AP axis extension, with key left-determining events, suggesting that Wnt3a functions as a trunk organizer. We are continuing to take molecular, genetic and cell biological approaches to examine the mechanisms underlying the control of LR asymmetric gene expression by Wnt3a. We are currently using conditional loss and gain of function alleles of beta-catenin to specifically examine its role in node and cilia function. We are also using microarray analyses to identify target genes of Wnt3a. Wnts and alternative signal transduction pathways in the regulation of cell polarity and AP axis elongationThe proper orientation and movement of cells in a three-dimensional framework defined by the body plan is critical for the formation, growth and elongation of the mammalian embryo. Wnts are known to regulate cell polarity and the orientation of mitotic cells and are thought to do so by signaling through alternative Wnt signaling pathways. Targeted mutations in Wnt5a reveal phenotypes distinct from Wnts that signal through beta-catenin. Wnt5a does not participate in AP axis specification but instead appears to be required for the polarized cell movements of convergent-extension. Convergent-extension refers to the lengthening and narrowing of a field of cells and is an important component of AP axis extension. Our current studies are aimed at understanding the cellular mechanisms underlying Wnt5a function and suggest that Wnt5a may be required for establishing cell polarity in the primitive streak. Genetic studies indicate that Wnt5a may regulate convergent extension by regulating both the canonical pathway as well as the Wnt/PCP pathway. The small GTPase RhoA is known to be a key regulator of cell polarity and morphogenesis and has been recently shown in frogs to be coupled to the Wnt pathway by a formin-homology protein called Daam. In collaboration with Xi He (Harvard Med), we have cloned two mouse homologs of Daam. Interestingly, Daam expression patterns are similar to that of Wnt5a, consistent with the Daam molecules signaling downstream of Wnt5a in the Wnt/PCP pathway. We have generated both deletions and conditional alleles of these genes, and have shown that mDaam1 is required for embryogenesis. Loss of mDaam1 leads to a preimplantation phenotype that is consistent with mDaam1 being required for the generation of apical-basal polarity in trophectoderm cells. Furthermore, we have demonstrated that mDaam1 interacts genetically with components of the Wnt/PCP pathway. Compound mutants lack heads or display holoprosencephaly, consistent with mDaam1 playing a role in regulating convergent-extension of midline cells required for head induction.